We present a closed-form joint probability distribution of disulfide-bonded cysteine-cysteine conformations in proteins. The model is inferred under a Bayesian framework and extends the dihedral conformation verification capabilities of PhiSiCal-Checkup [1], previously restricted to individual amino acid conformations, to now include disulfide-bonded cysteine pairs. It further permits rapid, on-demand analytical derivation of marginal and conditional distributions over any subset of dihedral conformational angles, enabling efficient probability evaluation and random sampling of disulfide conformations. These capabilities provide a unified probabilistic framework for downstream analyses, including modelling, refinement, simulation, and design. Software to analyze disulfide bonded cysteine conformations is accessible fromhttps://lcb.infotech.monash.edu/phisical/disulfide/.
Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (ncRNAs) that guide Piwi proteins to complementary RNA targets and trigger gene silencing through secondary 22G siRNA amplification. Accurate identification of piRNA target sites is therefore critical for understanding cellular gene silencing. Previously, we developed piRTarBase to catalog canonical piRNA-mRNA interactions using pirScan, but it was limited to mRNA targets and canonical binding rules. To address these limitations, we developed piRTarBase2, an updated database integrating CLASH-derived interactions processed by ChiRA. piRTarBase2 combined pirScan and RNAup to characterize both canonical and non-canonical binding sites across mRNAs and ncRNAs. It further integrates multiple lines of supporting evidence, including expression fold changes in prg-1 mutants, 22G-RNA signals, and cross-linking-induced mutation sites, for confidence evaluation of the piRNA binding sites. piRTarBase2 provides a comprehensive resource for exploring experimentally supported piRNA-target interactions and is available at https://cosbi7.ee.ncku.edu.tw/pirtarbase_v2/.
Physical processing offers opportunities to tailor dairy protein functionality, but useful conditions depend on the target function and matrix. This narrative review compares electric-field, pressure and ultrasound treatments through function-specific processing windows. In a 10% (m/v) whey protein concentrate dispersion, pulsed electric fields at 17-21 kV cm- 1 and 8-15 kJ L- 1 improved solubility and emulsion stability after drying. Other exposures weakened whey protein gels or increased casein peptide release despite reduced cellular uptake. Pressure and ultrasound similarly produced endpoint-specific gains. Soluble-protein recovery, function-limiting aggregation, casein colloidal stability and native activity provide operational markers for defining useful conditions and detecting functional loss. Most upper boundaries remain unmeasured, and a milk-protein processing window for induction-generated electric fields remains unresolved. Integrating delivered exposure, matrix composition and matched controls with final-product performance can guide the design of dairy protein ingredients.
The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a human ACE2-adapted virus highlights the need for antiviral strategies to control emerging HKU5-related merbecoviruses. With no specific antivirals approved for merbecoviruses, repurposing SARS-CoV-2 Mpro inhibitors is a promising strategy. However, their inhibitory activity and binding mechanisms against HKU5-CoV Mpro remain poorly understood. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro in its apo state (1.75 Å) and in complex with the approved covalent inhibitor nirmatrelvir (1.91 Å) and the non-covalent inhibitor ensitrelvir (1.55 Å). These structures reveal an induced-fit mechanism, in which the S2 loop containing Leu49 adopts an open conformation in the apo state and undergoes inhibitor-specific conformational changes upon inhibitor binding. These structures provided a foundation for characterizing HKU5-CoV-2 Mpro via modeling and molecular dynamics simulations, demonstrating conserved inhibitor-binding modes across both lineages. Consistent with this, biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 Mpro exhibited nearly identical enzyme kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 Mpro. Structural comparison of the HKU5-CoV-1 and SARS-CoV-2 Mpro revealed a conserved S1 subsite, alongside key variations: Met25 lining S1′, a flexible Leu49-containing S2 loop, and a Ser171-mediated expanded S4 cavity. Despite these variations, nirmatrelvir and ensitrelvir showed potent inhibitory activity, with comparable double-digit nanomolar IC50 values across all three Mpro proteins. Computational analyses indicated that HKU5-CoV-2 Mpro engages nirmatrelvir and ensitrelvir in binding modes comparable to HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition and providing a structural basis for developing pan-coronavirus antivirals targeting emerging merbecoviruses.
To achieve high-value utilization of Yunnan rubing cheese by-products, xanthine oxidase (XOD) inhibitory peptides were prepared from rubing cheese whey via enzymatic hydrolysis and ultrafiltration. The ultrafiltration fractions (molecular weight < 3 kDa) exhibited relatively high in vitro XOD inhibitory activity, non-toxicity (0.0625-1.5 mg mL(-1) in HepG2 cells), and good stability in acid, alkali, heat and gastrointestinal simulations. Two novel peptides, FPGPIP and WSPWQ, were identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with in-vitro and in-silico. Both peptides showed good hydrophobicity and XOD inhibitory activity. FPGPIP and WSPWQ, were selected with IC50 values of 0.93 mM and 0.88 mM. Fourier transform infrared (FTIR) spectroscopy analysis revealed that the secondary structures of FPGPIP and WSPWQ are mainly composed of beta-turn/beta-sheet conformations, which are crucial for the stability and activity of the peptides. Circular dichroism (CD) indicated that FPGPIP and WSPWQ can alter the secondary structure of XOD. Molecular docking demonstrated their binding to XOD's active sites through hydrogen bonding and hydrophobic interaction. Molecular dynamics analysis further showed that both peptides FPGPIP and WSPWQ could form stable complexes with XOD, thus exerting inhibitory effects and showing good stability. Network pharmacology further predicts that these peptides may exert uric acid-lowering effects through synergistic multi-target and multi-pathway mechanisms. This study demonstrates rubing cheese whey's potential as a sustainable source of uric acid-lowering peptides, offering insights for developing functional ingredients from dairy by-products.
